This invention relates to a DNA fragment containing a gene which encodes the function of stabilizing a plasmid in a host microorganism. More specifically, it relates to a DNA fragment which contains a gene encoding a stabilizing function, and is derived from plasmid pBY503 obtained from Brevibacterium stationis IF012144, and to its use.
Coryneform bacteria including bacteria of the genus Brevibacterium are industrially useful microorganisms which produce amino acids, organic acids and purine nucleotide. The molecular breeding of Coryneform bacterial strains by the introduction of the recombinant DNA technology is still behind that of Escherichia coli strains. Particularly, it is strongly desired to develop industrially useful vectors of excellent stability using Coryneform bacteria as hosts.
Generally, with regard to the stability of constructed plasmids in hosts, various cases of genetic instability, such as segregating of a plasmid from a host during cultivation or deletion of an inserted gene, have been reported, and countermeasures against it have been considered.
For example, there was proposed a method of stabilizing the properties of a microorganism containing a plasmid, in which a plasmid having inserted thereinto a chromosomal gene DNA fragment coding for the property of not depending upon streptomycin derived from Escherichia coli is included in a streptomycin-dependent mutant of the genus Escherichia (Japanese Laid-Open Patent Publication No. 156591/1980). This method, however, is economically disadvantageous. Furthermore, since it is necessary to insert complex functions into the plasmid, it is foreseen that the plasmid will have difficulty in being distributed stably to daughter cells at the time of fission and proliferation of the host. Accordingly, it would encounter various problems before it could be successfully applied industrially.
Most of commercially useful natural plasmids obtained from Coryneform bacteria including bacteria of the genus Brevibacterium do not have a drug-resistant gene which can be a plasmid marker and are devoid of cloning sites to which useful genes can be bound. Accordingly, although these natural plasmids have a possibility of genetically improving bacteria of this genus, they are unsuitable for molecular breeding of Coryneform bacteria.
The present inventors previously developed industrially useful plasmid vectors such as pCRY2 and pCRY3 by introducing a drug-resistant marker, cloning sites, and a gene region necessary for replication in Escherichia coli into natural plasmid pBY502 or pBY503 (see Japanese Laid-Open Patent Publication No. 191686/1989). However, these plasmid vectors show considerable instability in bacterial cells and are not fully efficient in producing gene products in large quantities within the cells.
It is desired therefore to develop a method of stabilizing a plasmid, in which the plasmid is accurately distributed through generations from parent cells to daughter cells within cells of a transformed microorganism.